Protein synthesis encompasses the processes of translation initiation, elongation, and termination, each of which has evolved to occur with great accuracy and has the capacity to be a regulated step in the pathway of gene expression. Recent studies, including those suggesting that events at termination may regulate the ability of ribosomes to recycle to the start site of the same mRNA, have underscored the potential of termination to regulate other aspects of translation. The RNA triplets UAA, UAG, and UGA are non-coding and promote translational termination. Termination starts when one of the three termination codons enters the A site of the ribosome, thereby signaling the polypeptide chain release factors to bind and recognize the termination signal. Subsequently, the ester bond between the 3′ nucleotide of the transfer RNA (“tRNA”) located in the ribosome's P site and the nascent polypeptide chain is hydrolyzed, the completed polypeptide chain is released, and the ribosome subunits are recycled for another round of translation.
Nonsense-mediated mRNA decay is a surveillance mechanism that minimizes the translation and regulates the stability of RNAs that contain chain termination nonsense mutations (see, e.g., Hentze & Kulozik, 1999, Cell 96:307-310; Culbertson, 1999, Trends in Genetics 15:74-80; Li & Wilkinson, 1998, Immunity 8:135-141; and Ruiz-Echevarria et al., 1996, Trends in Biological Sciences, 21:433-438). Chain termination nonsense mutations are caused when a base substitution or frameshift mutation changes a codon into a termination codon, i.e., a premature stop codon that causes translational termination. In nonsense-mediated mRNA decay, mRNAs with premature stop codons are frequently subjected to degradation. A truncated protein is produced as a result of the translation apparatus prematurely terminating at the stop codon.
Nonsense mutations cause approximately 10 to 30 percent of the individual cases of virtually all inherited diseases. Although nonsense mutations inhibit the synthesis of a full-length protein to one percent or less of wild-type levels, minimally boosting the expression levels of the full-length protein to between five and fifteen percent of normal levels can eliminate or greatly reduce the severity of disease. Nonsense suppression causes the read-through of a termination codon and the generation of full-length protein. Certain aminoglycosides have been found to promote nonsense suppression (see, e.g. Bedwell et al., 1997, Nat. Med. 3:1280-1284 and Howard et al., 1996, Nat. Med. 2:467-469). Clinical approaches that target the translation termination event to promote nonsense suppression have recently been described for model systems of cystic fibrosis and muscular dystrophy; gentamicin is an aminoglycoside antibiotic that causes translational misreading and allows the insertion of an amino acid at the site of the nonsense codon in models of cystic fibrosis, Hurlers Syndrome, and muscular dystrophy (see, e.g., Barton-Davis et al., 1999, J. Clin. Invest. 104:375-381). These results strongly suggest that drugs that promote nonsense suppression by altering translation termination efficiency of a premature termination codon can be therapeutically valuable in the treatment of diseases caused by nonsense mutations.
Certain classes of known antibiotics have been characterized and found to interact with RNA. For example, the antibiotic thiostrepton binds tightly to a 60-mer from ribosomal RNA (Cundliffe et al., 1990, in The Ribosome: Structure, Function & Evolution (Schlessinger et al., eds.) American Society for Microbiology, Washington, D.C. pp. 479-490), and bacterial resistance to various antibiotics often involves methylation at specific rRNA sites (Cundliffe, 1989, Ann. Rev. Microbiol. 43:207-233). In addition, certain aminoglycosides and other protein synthesis inhibitors have been found to interact with specific bases in 16S rRNA (Woodcock et al., 1991, EMBO J. 10:3099-3103); moreover, an oligonucleotide analog of the 16S rRNA has been shown to interact with certain aminoglycosides (Purohit et al., 1994, Nature 370:659-662). Aminoglycosidic aminocyclitol (aminoglycoside) antibiotics and peptide antibiotics are known to inhibit group I intron splicing by binding to specific regions of the RNA (von Ahsen et al., 1991, Nature (London) 353:368-370). Some of these same aminoglycosides have also been found to inhibit hammerhead ribozyme function (Stage et al., 1995, RNA 1:95-101). A molecular basis for hypersensitivity to aminoglycosides has been found to be located in a single base change in mitochondrial rRNA (Hutchin et al., 1993, Nucleic Acids Res. 21:4174-4179). Aminoglycosides have also been shown to inhibit the interaction between specific structural RNA motifs and the corresponding RNA binding protein. Zapp et al. (Cell, 1993, 74:969-978) has demonstrated that the aminoglycosides neomycin B, lividomycin A, and tobramycin can block the binding of Rev, a viral regulatory protein required for viral gene expression, to its viral recognition element in the IIB (or RRE) region of HIV RNA. This blockage appears to be the result of competitive binding of the antibiotics directly to the RRE RNA structural motif.
Citation or identification of any reference in Section 2 of this application is not an admission that such reference is available as prior art to the present invention.